SAM operon

ABSTRACT

The invention provides isolated nucleic acid compounds encoding a novel SAM synthetase of  Streptomyces fradiae.  Also provided are vectors and transformed heterologous host cells for expressing the SAM synthetase and a method for preparing S-adenosylmethionine from recombinant host cells transformed with the SAM synthetase gene.

[0001] We hereby claim the benefit under Title 35, United States Code, §119(e) of U.S. provisional patent application No. 60/030,898 filed Nov. 13, 1996

BACKGROUND OF THE INVENTION

[0002] This invention relates to recombinant DNA technology. In particular the invention pertains to the cloning of the SAM operon genes from Streptomyces fradiae and the use of said genes and their encoded proteins to produce S-adenosylmethionine (SAM) in a recombinant host.

[0003] S-adenosylmethionine is a product of natural origin found in all living organisms. SAM is a product of considerable importance for its role in biological reactions such as transmethylations. While the enzymes that catalyze these reactions are varied in their substrate specificity they are practically universal in their requirement of S-adenosylmethionine as the ultimate methyl group donor. Some methyl transfer reactions are important in the synthesis of certain antibiotics, such as tylosin.

[0004] Tylosin is a macrolide antibiotic composed of a 16-membered branched lactone, tylactone, and residues of three attached sugars, mycaminose, mycarose, and mycinose. Tylosin is produced commercially by Streptomyces fradiae (ATCC 19609; NRRL 2702) and is used as an animal growth promotant and veterinary antibiotic. The multi-step biosynthesis of tylosin has been studied both physiologically and genetically (See generally, R. H. Baltz and E. T. Seno, “Genetics of Streptomyces fradiae and tylosin biosynthesis. Ann. Rev. Microbiol. 42, 547-74 (1988)). At least 13 biosynthetic genes and 2 regulatory genes are necessary for normal production of tylosin. Tylosin synthesis requires multiple methylation reactions, the last two of which are rate-limiting. In the last step a specific methyltransferase catalyzes the transfer of a methyl group from SAM to the tylosin precursor molecule, macrocin. Thus, the availability of SAM as the methyl group donor is essential in the synthesis of tylosin.

[0005] S-adenosylmethionine is produced when an adenosyl group is transferred from ATP to methionine. SAM is synthesized in the cell by the action of three enzymes encoded by the SAM operon—SAM synthetase, methyl transferase (MT), and methylene tetrahydrofolate reductase (MTHR).

[0006] SUMMARY OF INVENTION

[0007] The present invention provides, inter alia, isolated nucleic acid molecules comprising the SAM operon from Streptomiyces fradiae. The invention also provides the protein products encoded by the SAM operon, in substantially purified form.

[0008] Having the cloned SAM operon of Streptomyces fradiae enables the production of S-adenosylmethionine in recombinant host cells.

[0009] In one embodiment the present invention relates to an isolated nucleic acid that encodes SAM synthetase from Streptomiyces fradiae, said nucleic acid comprising nucleotide residues 986 through 2209 of the nucleotide sequence identified as SEQ ID NO. 1.

[0010] In another embodiment the present invention relates to an isolated nucleic acid that encodes MT from Streptomyces fradiae, said nucleic acid comprising nucleotide residues 2241 through 3341 of SEQ ID NO.1.

[0011] In another embodiment the present invention relates to an isolated nucleic acid that encodes MTHR from Streptomyces fradiae, said nucleic acid comprising nucleotide residues 3338 through 4255 of SEQ ID NO.1.

[0012] In another embodiment the present invention relates to a novel SAM synthetase from Sreptomyces fradiae in substantially purified form comprising the sequence identified as SEQ ID NO. 2.

[0013] In still another embodiment the present invention relates to a novel MT from Streptomyces fradiae in substantially purified form comprising the sequence identified as SEQ ID NO. 3.

[0014] In yet another embodiment the present invention relates to a novel MTHR from Streptomyces fradiae in substantially purified form comprising the sequence identified as SEQ ID NO.5.

[0015] In a further embodiment the present invention relates to a ribonucleic acid molecule encoding SAM synthetase, said ribonucleic acid molecule comprising residues 986 through 2209 of the sequence identified as SEQ ID NO. 6:

[0016] In yet another embodiment, the present invention relates to a recombinant DNA vector that incorporates the Streptomyces fradiae SAM operon genes in operable linkage to gene expression sequences enabling said genes to be transcribed and translated in a host cell.

[0017] In still another embodiment the present invention relates to homologous or heterologous host cells which have been transformed or transfected with one or more of the cloned SAM operon genes from Streptomyces fradiae such that said gene(s) is/are expressed in the host cell.

[0018] In a still further embodiment, the present invention relates to a method for producing S-adenosylmethionine in recombinant host cells transformed with the S. fradiae SAM synthetase gene.

DESCRIPTION OF THE DRAWING

[0019]FIG. 1. Plasmid pRBD26, useful for expression of the Streptomyces fradiae SAM synthetase gene of the present invention in the homologous host cell or other actinomycete.

[0020]FIG. 2. Activated methyl cycle.

DEFINITIONS

[0021] The term “operon” as used herein refers to a genetic unit comprising a region of a chromosome having one or more structural genes said unit producing a messenger RNA molecule that may or may not be polycistronic (i.e. encoding more than one protein). Transcription of said RNA is under the control of a single promoter.

[0022] “SAM” refers to S-adenosylmethionine.

[0023] “MT” refers to methyltransferase.

[0024] “THF” refers to tetrahydrofolate.

[0025] “MTHR” refers to methylene tetrahydrofolate reductase.

[0026] “ATP” refers to adenosine triphosphate.

[0027] The terms “cleavage” or “restriction” of DNA refers to the catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA (viz. sequence-specific endonucleases). The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements are used in the manner well known to one of ordinary skill in the art. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer or can readily be found in the literature.

[0028] The term “plasmid” refers to an extrachromosomal genetic element. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accordance with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

[0029] “Recombinant DNA cloning vector” as used herein refers to any autonomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.

[0030] The term “recombinant DNA expression vector” as used herein refers to any recombinant DNA cloning vector, for example a plasmid or phage, in which a promoter and other regulatory elements are present to enable transcription of the inserted DNA.

[0031] The term “vector” as used herein refers to a nucleic acid compound used for introducing exogenous DNA into host cells. A vector comprises a nucleotide sequence which may encode one or more protein molecules. Plasmids, cosmids, viruses, and bacteriophages, in the natural state or which have undergone recombinant engineering, are examples of commonly used vectors.

[0032] The terms “complementary” or “complementarity” as used herein refers to the capacity of purine and pyrimidine nucleotides to associate through hydrogen bonding in double stranded nucleic acid molecules. The following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil.

[0033] “Isolated nucleic acid compound” refers to any RNA or DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location.

[0034] A “primer” is a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation of, for example, a nucleic acid molecule.

[0035] The term “promoter” refers to a DNA sequence which directs transcription of DNA to RNA.

[0036] A “probe” as used herein is a labeled nucleic acid compound which hybridizes with a complementary nucleic acid compound.

[0037] The term “hybridization” as used herein refers to a process in which a single-stranded nucleic acid molecule joins with a complementary strand through nucleotide base pairing. “Selective hybridization” refers to hybridization under conditions of high stringency. The degree of hybridization depends upon, for example, the degree of complementarity, the stringency of hybridization, and the length of hybridizing strands.

[0038] The term “stringency” refers to hybridization conditions. High stringency conditions disfavor non-homologous basepairing. Low stringency conditions have the opposite effect. Stringency may be altered, for example, by temperature and salt concentration.

DETAILED DESCRIPTION

[0039] The SAM operon of the present invention comprises three genes encoding SAM synthetase, MT, and MTHR. Together these enzymes comprise the so-called “activated methyl cycle” which produces S-adenosylmethionine (see FIG. 2). The “activated methyl cycle” provides the methyl groups required for the final steps in tylosin production.

[0040] The SAM synthetase gene disclosed herein comprises a DNA sequence of 1224 nucleotide base pairs (residues 986 through 2209 of SEQ ID NO. 1). The MT gene disclosed herein comprises a DNA sequence of 1101 nucleotide base pairs (residues 2241 through 3341 of SEQ ID NO.1). The MTHR gene disclosed herein comprises a DNA sequence of 918 nucleotide base pairs (residues 3338 through 4255 of SEQ ID NO.4). There are no intervening sequences in the SAM operon. The 5′ end of the MTHR gene overlaps (in another reading frame) with four nucleotide residues at the 3′ end of the MT gene. Specifically, the “TGA” stop codon of the MT gene ends at residue 3341 of SEQ ID NO.4, while the “GTG” start codon of the MTHR gene begins at residue 3338 of SEQ ID NO.4. Those skilled in the art will recognize that owing to the degeneracy of the genetic code (i.e. 64 codons which encode 20 amino acids), numerous “silent” substitutions of nucleotide base pairs could be introduced into this sequence without altering the identity of the encoded amino acid(s) or protein product. All such substitutions are intended to be within the scope of the invention.

[0041] The SAM synthetase of the present invention, designated SEQ ID NO.2, comprises a protein of 407 amino acid residues. The MT of the present invention, designated SEQ ID NO.3, comprises a protein of 366 amino acid residues. The MTHR of the present invention, designated SEQ ID NO.5, comprises a protein of 305 amino acid residues.

[0042] Gene Isolation Procedures

[0043] Those skilled in the art will recogize that the gene of the present invention may be obtained by a plurality of applicable genetic and recombinant DNA techniques including, for example, polymerase chain reaction (PCR) amplification, or de novo DNA synthesis.(See e.g., J. Sambrook et al. Molecular Cloning, 2d Ed. Chap. 14 (1989)).

[0044] Methods for constructing gene libraries in a suitable vector such as a plasmid or phage for propagation in procaryotic or eucaryotic cells are well known to those skilled in the art. [See e.g. J. Sambrook et al. Supra]. Suitable cloning vectors are widely available.

[0045] Skilled artisans will recognize that the SAM operon genes of Streptomyces fradiae comprising the present invention or fragments thereof could be isolated by PCR amplification of Streptomyces fradiae genomic DNA or cDNA using oligonucleotide primers targeted to a suitable region of SEQ ID NO. 1. The coding regions of the MT and MTHR genes are, respectively, 2241 through 3341, and 3338 through 4255 of SEQ ID NO.1. Methods for PCR amplification are widely known in the art. See e.g. PCR Protocols: A Guide to Method and Application, Ed. M. Innis et al., Academic Press (1990). The amplification reaction comprises genomic DNA, suitable enzymes, primers, and buffers, and is conveniently carried out in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, Conn.). Amplification of a DNA fragment of the correct size can be detected most conveniently by agarose gel electrophoresis.

[0046] Protein Production Methods

[0047] One embodiment of the present invention relates to the substantially purified SAM operon proteins or fragments thereof encoded by the genes disclosed herein.

[0048] Skilled artisans will recognize that the proteins of the present invention can be synthesized by any number of different methods. The amino acid compounds of the invention can be made by chemical methods well known in the art, including solid phase peptide synthesis or recombinant methods. Both methods are described in U.S. Pat. No. 4,617,149, incorporated herein by reference.

[0049] The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts in the area. See, e.g., H. Dugas and C. Penney, Bioorganic Chemistry (1981) Springer-Verlag, New York, 54-92. For example, peptides may be synthesized by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Foster City, Calif.) and synthesis cycles supplied by Applied Biosystems. Protected amino acids, such as t-butoxycarbonyl-protected amino acids, and other reagents are commercially available from many chemical supply houses.

[0050] The proteins of the present invention can also be produced by recombinant DNA methods using the cloned SAM operon genes of Streptomyces fradiae disclosed herein. Recombinant methods are preferred if a high yield is desired. Expression of said cloned genes can be carried out in a variety of suitable host cells well known to those skilled in the art. In a recombinant method, a gene is introduced into a host cell by any suitable means, well known to those skilled in the art. While chromosomal integration of the cloned SAM operon genes is within the scope of the present invention, it is preferred that the genes be cloned into a suitable extra-chromosomally maintained expression vector, the coding region of the genes in operable linkage to a constitutive or inducible promoter.

[0051] The basic steps in the recombinant production of the SAM operon enzymes, SAM synthetase, MT, or MTHR of the present invention are:

[0052] a) constructing a natural, synthetic or semi-synthetic DNA encoding said enzyme(s);

[0053] b) integrating said DNA into an expression vector in a manner suitable for expressing said enzyme(s), as the natural protein product or as a fusion protein;

[0054] c) transforming or otherwise introducing said vector into an appropriate eucaryotic or prokaryotic host cell forming a recombinant host cell,

[0055] d) culturing said recombinant host cell in a manner to express said enzyme(s); and

[0056] e) recovering and substantially purifying said enzyme(s) by any suitable means, well known to those skilled in the art.

[0057] Expressing Recombinant S. fradiae SAM Operon in a Procaryotic or Eucaryotic Host Cell

[0058] In general, procaryotes are used for cloning DNA sequences and for constructing the vectors of the present invention. Procaryotes may also be employed in the production of the protein of the present invention. For example, the Escherichia coli K12 strain 294 (ATCC No. 31446) is particularly useful for the prokaryotic expression of foreign proteins. Other strains of E. coli, bacilli such as Bacillus subtilis, enterobacteriaceae such as Salmonella typhimurium or Serratia marcescans, various Pseudomonas species and Actinomycetes, such as Streptomyces fradiae , Streptomyces coelicolor, and Streptomyces lividans, may also be employed as host cells in the cloning and expression of the recombinant proteins of this invention.

[0059] Promoter sequences suitable for driving the expression of genes in procaryotes include b-lactamase [e.g. vector pGX2907, ATCC 39344, contains a replicon and b-lactamase gene], lactose systems [Chang et al., Nature (London), 275:615 (1978); Goeddel et al., Nature (London), 281:544 (1979)], alkaline phosphatase, and the tryptophan (trp) promoter system [vector pATH1 (ATCC 37695) which is designed to facilitate expression of an open reading frame as a trpE fusion protein under the control of the trp promoter]. Hybrid promoters such as the tac promoter (isolatable from plasmid pDR540, ATCC-37282) are also suitable. Useful promoters for driving gene expression in a Streptomyces host are known. For example, the snpR promoter is useful for gene expression in Streptomyces (See e.g. 6th Conference on the Genetics and Molecular Biology of Industrial Microorganisms, Oct. 20-24, 1996, Bloomington, Ind., Abstract P37). Still other bacterial promoters, whose nucleotide sequences are generally known, enable one of skill in the art to ligate such promoter sequences to DNA encoding the proteins of the instant invention using linkers or adapters to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dalgarno sequence operably linked to the DNA encoding the desired polypeptides. The cloned DNA comprising the SAM operon of the present invention carries the endogenous promoter in the region comprising nucleotide residues 1 through 985 of SEQ ID NO.1. This promoter will function in other Actinomycetes. These examples are illustrative rather than limiting.

[0060] The protein of this invention may be synthesized either by direct expression or as a fusion protein comprising the protein of interest as a translational fusion with another protein or peptide which may be removable by enzymatic or chemical cleavage. Expression as a fusion protein may prolong the half-life, increase the yield of the desired peptide, or provide a convenient means of purifying the protein. A variety of peptidases (e.g. enterokinase and thrombin) cleave a polypeptide at specific sites or digest peptides from the amino or carboxy termini (e.g. diaminopeptidase). Furthermore, particular chemicals (e.g. cyanogen bromide) will cleave a polypeptide chain at specific sites. The skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semi-synthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites. See e.g., P. Carter, “Site Specific Proteolysis of Fusion Proteins”, Chapter 13, in Protein Purification: From Molecular Mechanisms to Large Scale Processes, American Chemical Society, Washington, D.C. (1990).

[0061] In addition to procaryotes, a variety of mammalian cell systems and eucaryotic microorganisms such as yeast are suitable host cells. The yeast Saccharomyces cerevisiae is the most commonly used eucaryotic microorganism. A number of other yeasts such as Kluyveromyces lactis are also suitable. For expression in Saccharomyces, the plasmid YRp7 (ATCC-40053), for example, may be used. See, e.g., L. Stinchcomb, et al., Nature, 282:39 (1979); J. Kingsman et al., Gene, 7:141 (1979); S. Tschemper et al., Gene, 10:157 (1980). Plasmid YRp7 contains the TRP1 gene which provides a selectable marker for use in a trp1 auxotrophic mutant.

[0062] Purification of Recombinantly-Produced SAM Operon Enzymes

[0063] An expression vector carrying any of the cloned SAM operon genes as claimed herein is transformed or transfected into a suitable host cell using standard methods. Cells which contain the vector are propagated under conditions suitable for expression of the SAM operon enzyme(s) encoded by the vector. As an example, a vector-bound SAM synthetase gene is placed under the control of an inducible promoter. Suitable growth conditions would incorporate an appropriate inducer. Recombinantly-produced SAM synthetase may then be purified from cellular extracts of transformed cells by any suitable means, well known to those skilled in the art.

[0064] In a preferred process for protein purification the gene encoding the SAM operon enzyme of the present invention is modified at the 5′ end to incorporate several histidine residues at the amino terminus of the encoded protein product. The “His-tag” enables a single-step protein purification method referred to as “immobilized metal ion affinity chromatography” (IMAC), essentially as described in U.S. Pat. No. 4,569,794 which hereby is incorporated by reference. The IMAC method enables rapid isolation of substantially pure protein starting from a crude cellular extract.

[0065] Other embodiments of the present invention comprise isolated nucleic acid sequences which encode SEQ ID NO:2, SEQ ID NO.3, and SEQ ID NO.5. As skilled artisans will recognize, the amino acid compounds of the invention can be encoded by a multitude of different nucleic acid sequences because most of the amino acids are encoded by more than one codon due to the degeneracy of the genetic code. Because these alternative nucleic acid sequences would encode the same amino acid sequences, the present invention further comprises these alternate nucleic acid sequences.

[0066] Nucleic acid sequences encoding the proteins of the invention may be produced using synthetic methodology. The synthesis of nucleic acids is well known in the art. See, e.g., E. L. Brown, R. Belagaje, M. J. Ryan, and H. G. Khorana, Methods in Enzymology, 68:109-151 (1979). The DNA segments could be generated using a conventional DNA synthesizing apparatus, such as the Applied Biosystems Model 380A or 380B DNA synthesizers (Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, Calif. 94404) which employ phosphoramidite chemistry. Alternatively, phosphotriester chemistry may be employed to synthesize the nucleic acids of this invention. [See, e.g., M. J. Gait, ed., Oligonucleotide Synthesis, A Practical Approach, (1984).]

[0067] In an alternative and preferred methodology, namely PCR, the DNA sequence comprising a portion or all of SEQ ID NO:1 can be generated from Streptomyces fradiae genomic DNA using suitable oligonucleotide primers complementary to SEQ ID NO:1 or region therein, as described in U.S. Pat. No. 4,889,818, which hereby is incorporated by reference. Suitable protocols for performing the PCR are widely known and are disclosed in, for example, PCR Protocols: A Guide to Method and Applications, Ed. Michael A. Innis et al., Academic Press, Inc. (1990).

[0068] The ribonucleic acids of the present invention may be prepared using the polynucleotide synthetic methods discussed supra, or they may be prepared enzymatically using RNA polymerase to transcribe a suitable DNA template.

[0069] The most preferred systems for preparing the ribonucleic acids of the present invention employ the RNA polymerase from the bacteriophage T7 or the bacteriophage SP6. These RNA polymerases are highly specific, requiring the insertion of bacteriophage-specific sequences at the 5′ end of the template to be transcribed. See, J. Sambrook, et al., supra, at 18.82-18.84.

[0070] This invention also provides nucleic acids, RNA or DNA, which are complementary to SEQ ID NO:1 or SEQ ID NO:6.

[0071] The present invention also provides probes and primers useful for a variety of molecular biology techniques including, for example, hybridization screens of genomic or subgenomic libraries. A nucleic acid compound comprising SEQ ID NO:1, SEQ ID NO:6 or a complementary sequence thereof, or a fragment thereof, and which is at least 18 base pairs in length, and which will selectively hybridize to Streptomiyces fradiae DNA or mRNA encoding the SAM operon of the present invention, is provided. Preferably, the 18 or more base pair compound is DNA. These probes and primers can be prepared by enzymatic methods well known to those skilled in the art (See e.g. Sambrook et al. supra). In a most preferred embodiment these probes and primers are synthesized using chemical means as described above.

[0072] Another aspect of the present invention relates to recombinant DNA cloning vectors and expression vectors comprising the nucleic acids of the present invention. Many of the vectors encompassed within this invention are described above. The preferred nucleic acid vectors are those which comprise DNA. The most preferred recombinant DNA vectors comprise the isolated DNA sequence, SEQ ID NO:1 or suitable region therof. Plasmid pRBD26 is an especially preferred DNA vector of the present invention.

[0073] The skilled artisan understands that choosing the most appropriate cloning vector or expression vector depends upon a number of factors including the availability of restriction enzyme sites, the type of host cell into which the vector is to be transfected or transformed, the purpose of the transfection or transformation (e.g., stable transformation as an extrachromosomal element, or integration into the host chromosome), the presence or absence of readily assayable or selectable markers (e.g., antibiotic resistance and metabolic markers of one type and another), and the number of copies of the gene to be present in the host cell.

[0074] Vectors suitable to carry the nucleic acids of the present invention comprise RNA viruses, DNA viruses, lytic bacteriophages, lysogenic bacteriophages, stable bacteriophages, plasmids, viroids, and the like. The most preferred vectors are plasmids.

[0075] When preparing an expression vector the skilled artisan understands that there are many variables to be considered, for example, whether to use a constitutive or inducible promoter. Inducible promoters are preferred because they enable high level, regulatable expression of the operably-linked genes of the present invention. The skilled artisan will recognize a number of inducible promoters which respond to a variety of inducers, for example, carbon source, metal ions, heat, and others. The practitioner also understands that the amount of nucleic acid or protein to be produced dictates, in part, the selection of the expression system. The addition of certain nucleotide sequences is useful for directing the localization of a recombinant protein. For example, a sequence encoding a signal peptide preceding the coding region of a gene, is useful for directing extra-cellular export of the resulting polypeptide.

[0076] Host cells harboring the nucleic acids disclosed herein are also provided by the present invention. A preferred host is E. coli which has been transfected or transformed with a vector which comprises a nucleic acid of the present invention. Another preferred host is any member of the Actinomycetes.

[0077] The present invention also provides a method for constructing a recombinant host cell capable of expressing SEQ ID NO:2, SEQ ID NO.3, and/or SEQ ID NO.5, said method comprising transforming or otherwise introducing into a host cell a recombinant DNA vector that comprises an isolated DNA sequence which encodes said SEQs or fragments thereof. Preferred vectors for expression are those which comprise SEQ ID NO:1. An especially preferred expression vector for use in E. coli is pRBD26, which comprises SEQ ID NO:1. (See FIG. 1). Transformed host cells may be cultured under conditions well known to skilled artisans such that the vector-encoded SAM operon enzymes are expressed in the recombinant host cell.

[0078] Regulating Production of Tylosin and Other Methylated Compounds

[0079] The SAM operon maps within the tylosin biosynthetic gene cluster, a region of the S. fradiae chromosome that encodes the structural and regulatory genes needed for tylosin biosynthesis. The SAM operon enzymes comprise the so-called “activated methyl cycle.” This series of enzymatic reactions produces the methyl group donor molecule, SAM. The enzymatic activities encoded by the SAM operon as well as the chromosomal location of the SAM operon suggests that the SAM operon may participate in the regulation of tylosin synthesis within the cell. Thus, controlling the expression of the SAM operon genes may lead to controlled expression and production of certain methylated compounds within the cell, such as tylosin or other methylated antibiotic. Altering the levels of production of methylated compounds might be possible by altering the copy number and/or expression of the SAM operon genes.

[0080] The following examples more fully describe the present invention. Those skilled in the art will recognize that the particular reagents, equipment, and procedures described below are merely illustrative and are not intended to limit the present invention in any manner.

EXAMPLE 1 Construction of a DNA Vector Carrying the Streptomyces fradiae SAM Operon Genes

[0081] Plasmid pRBD26 (See FIG. 1) is an approximately 10.3 kilobase pair vector which can be propagated in E. coli and used for targeted integration of the SAM operon into the S. fradiae chromosome. This plasmid contains an origin of replication (OriT), an ampicillin resistance gene (Amp), the intØC31 site, useful for targeting integration into the bacteriophage ØC31 attachment site on the S. fradiae chromosome.

[0082] Plasmid pRBD26 carries a 4.8 kilobase pair fragment of S. fradiae genomic DNA (SEQ ID NO.1) that encodes the SAM operon. The S. fradiae genomic DNA was ligated into the blunt-ended EcoRV site of pSET152 (Bierman et al. Gene, 116, 43-49, 1992) using standard cloning methods. The resulting plasmid was designated pRBD26.

EXAMPLE 2

[0083] Construction of a Vector for Expressing the Streptomyces fradiae SAM Synthetase Gene in a Heterologous Host

[0084] The DNA sequence coding for S. fradiae SAM synthetase is isolated from S. fradiae genomic DNA or from pRBD26, most conveniently by PCR using oligonucleotide primers complementary to the ₅′ and 3′ terminal regions of the gene (viz. nucleotide residues 986 through 2209 of SEQ ID NO.1). For ease of cloning the SAM synthetase gene, the oligonucleotide primers are synthesized to contain one or more restriction enzyme cloning sites. Also for convenience in purifying the encoded SAM synthetase protein, the SAM synthetase gene is modified at the 5′ end (viz. amino terminus of encoded protein) by adding an oligonucleotide encoding 8 histidine residues and a factor Xa cleavage site after the ATG start codon at nucleotide positions 988 of SEQ ID NO: 1. Placement of the histidine residues at the amino terminus of the encoded SAM synthetase protein enables the IMAC one-step protein purification procedure (See below).

[0085] The PCR amplified SAM synthetase gene is then inserted into an appropriate expression vector in which the SAM synthetase gene is operably-linked with a high expression promoter, for example the T7 promoter or the lambda pL promoter (See e.g. U.S. Pat. No. 4,874,703, incorporated by reference). Any suitable plasmid may be used for this purpose. A particularly useful plasmid for this purpose is pET11A, which is available commercially from Novogen (Madison, Wis.).

EXAMPLE 3

[0086] Expression of SAM Synthetase Gene in Echerichia coli

[0087] A plasmid capable of expressing SAM synthetase (see e.g. Example 2) is transformed into E. Coli BL21 (DE3) (hsdS gal lcIts857 ind1Sam7nin5lacUV5-T7gene 1) using standard methods (See e.g. Sambrook et al. Supra).

EXAMPLE 4

[0088] Purification of SAM Synthetase

[0089] Transformants selected as in Example 3 are chosen at random and tested for the presence of the transforming vector by agarose gel electrophoresis using quick plasmid preparations. Id. Colonies that contain the vector are grown, processed, and the SAM synthetase, produced by the vector-bound SAM synthetase gene, purified by immobilized metal ion affinity chromatography (IMAC), essentially as described in U.S. Pat. No. 4,569,794, the entire contents of which is hereby incorporated by reference.

[0090] Briefly, the IMAC column is prepared as follows. A metal-free chelating resin (e.g. SEPHAROSE 6B IDA, Pharmacia) is washed in distilled water to remove preservative substances and infused with a suitable metal ion [e.g. Ni(II), Co(II), or Cu(II)] by adding a 50 mM metal chloride or metal sulfate aqueous solution until about 75% of the interstitial spaces of the resin are saturated with colored metal ion. The column is then ready to receive a crude cellular extract prepared from a recombinant host transformed or transfected with a vector encoding the SAM synthetase of this invention.

[0091] After washing the column with a suitable buffer, pH 7.5 to remove unbound proteins and other materials, the bound protein is eluted in a buffer at pH 4.3, essentially as described in U.S. Pat. No.4,569,794.

EXAMPLE 5

[0092] Production of S-adenosylmethionine in a Recombinant E.coli Host Cell that Overexpresses the S. fradiae SAM Synthetase Gene

[0093] A recombinant vector carrying the S. fradiae SAM synthetase gene in operable linkage with a T7 promoter (see e.g. Example 2) is transformed or otherwise introduced into a suitable strain of E. coli. The recombinant cells are grown under conditions that induce gene expression from the vector-borne T7 promoter.

[0094] A crude extract is prepared from the induced culture by any suitable method and the extract contacted with an activated polysaccharide material, for example, as described in U.S. Pat. No. 4,028,183 the entire contents of which is incorporated by reference. The polysaccharide material is activated by a reagent suitable for bonding proteins, for example cyanogen bromide. This reaction is conveniently carried out in a column. A solution of ATP and methionine in a suitable buffer is passed through the column and the eluate, which is enriched in SAM, is collected. The SAM is precipitated with picrolonic acid (See e.g. Anal. Biochem. 4, 16-28, 1971).

EXAMPLE 6 Expression of the S. Fradiae SAM Operon in a Recombinant Actinomycete

[0095] Plasmid pRBD26 is transformed into E. coli BL21 as in Example 3. Transformants harboring the plasmid are used for conjugal transfer of pRBD26 to Streptomyces spp. essentially as described in Bierman et al. “Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. ” Gene, 116, 43-49 (1992). Briefly, one ml of a frozen mycelial culture of S. fradiae was diluted into 9 ml of TS broth and grown for 18 h aerobically at 29_ C. The culture was homogenized and 2 ml was transferred into 18 ml of fresh TS broth and grown for 16 h at 29_ C. to obtain a late log-phase culture. This culture was homogenized and fragmented with ultrasound, and 1 ml was transferred to 9 ml TS broth. The culture was incubated aerobically at 37_ C. for 3 h. The mycelium was recovered by centrifugation, washed once in TS broth and resuspended in 2 ml TS broth (recipient culture). The E. coli donor was grown at 37_ C. overnight in TY broth plus 100 ug apramycin (Am)/ml, subcultured 1:100 and grown for 3 h at 37_ C. The cells were pelleted, washed once in TS broth and resuspended in 2 ml TS broth (donor culture). Equal volumes of the donor culture and ten-fold dilutions of the recipient culture were mixed, and 100 ul were plated to AS1 (Streptomyces medium) supplemented with 10 mM MgCl₂. Plates were incubated at 37_ C. for 16 h, and then covered with 3 ml to 4 ml of soft R2 agar containing 1.5 mg nalidixic acid and 1.5 mg of Am. Incubation at 37_ C. was continued for about a week to allow outgrowth of the exconjugants.

1 6 4848 base pairs nucleic acid single linear DNA (genomic) NO NO CDS 986..2209 CDS 2241..3341 1 TCATGACACC TCTGGTGAGG GGGGGCAGCT CAGCTCACCC CCGATCCGGA CACGCCGGGT 60 CCGCGGGGAG TCCCGCGGCA CCGCCGCTCG TCAACGCAGC GGGGCGAGCA CATGTCTTT 120 TCACATTTCT CCTTGGGGGG GCGAGGCCGG CGCCCGGCCG TCGCCTTTCG TGAATGCGG 180 GGAGCCGCAT CGCGAACGCG ATGCGGCTCC GGGGAGACTT CTCGAACCCG AGAAGGAAG 240 GGATTCGGCG ATACGGTTCC GGAATACCGC GGCTCGTCAC GAAGCCGGTT CCGGATCAG 300 GGTGCGGCCG TTCCAGGGCA CACCACGCCT GTTCCCCGGA GCGTGACAGG ACGCTCATC 360 GGGTGTCAAC GCACCATTCG ACGGAGGGAG TTGGGCCGCA CCGGCCGGAG GGGTCCGAC 420 AGGGGTTTCG GGCGGGATCG AGAAACACTC GAGAAGCGGC GGAAAACACC GTGCGGCTG 480 CCGGACCCAG GCGCCACCCG TTCTTCATCG GCTCTCCAGC CGGCCTTGAG CGCCCCGGC 540 GGCCACCACC CGCCGAAAAC CGGGACCAAG GATTCACCGG GTTCGTGACC GTATTGCGG 600 GTGGGATCCC GGGGATGGGA TGCCGGATGG ATTTCTGAGC AGGCGTCGCC CGGCAGCCG 660 CGGGCCGTGG CCGGAACCGG CCGGGCCGGT CGGCGGGGTG TGCGCGGTGC CGGCCGAAG 720 CGTGGGCCGG AGCGCGGACC CGAGCGCCGT GTCAGCGCCG TGTCAGGGCT TTGGGGGCG 780 GCCCTCACAT ACTCGACCCG CGTTCACGGA GAGGAAGGCA GGGAAGGGCC CGTCCGCAC 840 GCGACCGGTG CCGGCGCCCC CGCCCGATGC CCGGCGCCGC CCCACCGAGC CGGACGCAC 900 GGAGGTTCCC ACCGCCCGGC CGGACGCCGG CGTGCCGTTC AGCCGGTGCG CGGGCCGGC 960 CGAGATCTCA CACCACTGGA GAGCC ATG TCA CGT CGC CTG TTC ACC TCG GAG 1012 Met Ser Arg Arg Leu Phe Thr Ser Glu 1 5 TCC GTG ACC GAG GGC CAC CCC GAC AAG ATC GCC GAC CGG ATC AGC GAC 1060 Ser Val Thr Glu Gly His Pro Asp Lys Ile Ala Asp Arg Ile Ser Asp 10 15 20 25 ACC GTC CTC GAT GCC CTG CTC GCC CGG GAC CCG CGG GCC AGG GTC GCC 1108 Thr Val Leu Asp Ala Leu Leu Ala Arg Asp Pro Arg Ala Arg Val Ala 30 35 40 GTC GAG ACC CTG ATC ACC ACC GGT CAG GTC CAT ATC GCC GGC GAG GTC 1156 Val Glu Thr Leu Ile Thr Thr Gly Gln Val His Ile Ala Gly Glu Val 45 50 55 ACC ACC ACC GCG TAC GCG CCC ATC GCC CAA CTG GTG CGC GAC ACC GTC 1204 Thr Thr Thr Ala Tyr Ala Pro Ile Ala Gln Leu Val Arg Asp Thr Val 60 65 70 CTG TCC ATC GGC TAC GAC TCC TCG GCC AAG GGC TTC GAC GGC GCC TCG 1252 Leu Ser Ile Gly Tyr Asp Ser Ser Ala Lys Gly Phe Asp Gly Ala Ser 75 80 85 TGC GGG GTG TCG GTC TCC ATC GGC GCG CAG TCC CCG GAC ATC GCC CGG 1300 Cys Gly Val Ser Val Ser Ile Gly Ala Gln Ser Pro Asp Ile Ala Arg 90 95 100 105 GGT GTG GAC ACC GCG TAC GAG CGG CGG GGC GGG GGC ACG GCC CCG GGC 1348 Gly Val Asp Thr Ala Tyr Glu Arg Arg Gly Gly Gly Thr Ala Pro Gly 110 115 120 GGA CCG GGT GAC GAG CTG GAC CGG CAG GGC GCG GGC GAC CAG GGC CTG 1396 Gly Pro Gly Asp Glu Leu Asp Arg Gln Gly Ala Gly Asp Gln Gly Leu 125 130 135 ATG TTC GGC TAC GCC TGC GAC GAG ACC CCC GAG CTG ATG CCG CTG CCG 1444 Met Phe Gly Tyr Ala Cys Asp Glu Thr Pro Glu Leu Met Pro Leu Pro 140 145 150 ATC AAC CTG GCG CAC CGG CTC TCC CGG CGG CTG TCG GAG GTG CGG AAG 1492 Ile Asn Leu Ala His Arg Leu Ser Arg Arg Leu Ser Glu Val Arg Lys 155 160 165 AAC GGC ACG ATC CCC TAC CTC CGC CCC GAC GGC AAG ACC CAG GTC ACC 1540 Asn Gly Thr Ile Pro Tyr Leu Arg Pro Asp Gly Lys Thr Gln Val Thr 170 175 180 185 ATC GAG TAC GAC GGC GAC AAG GCG GTC CGC CTC GAC ACG GTC GTG GTC 1588 Ile Glu Tyr Asp Gly Asp Lys Ala Val Arg Leu Asp Thr Val Val Val 190 195 200 TCC TCC CAG CAC GCC TCC GGC ATC GAC CTG GAC TCG CTA CTG GCG CCC 1636 Ser Ser Gln His Ala Ser Gly Ile Asp Leu Asp Ser Leu Leu Ala Pro 205 210 215 GAC ATC CGC CGG CAT GTC GTG GAG CCC GTC CTC GCC GGA CTG GCC GAG 1684 Asp Ile Arg Arg His Val Val Glu Pro Val Leu Ala Gly Leu Ala Glu 220 225 230 GAC GGC ATC AAG CTG GAC ACC GCG GGC TAC CGG CTG CTG GTC AAT CCC 1732 Asp Gly Ile Lys Leu Asp Thr Ala Gly Tyr Arg Leu Leu Val Asn Pro 235 240 245 ACC GGC CGG TTC GAG ATC GGC GGC CCG ATG GGC GAC GCG GGC CTG ACC 1780 Thr Gly Arg Phe Glu Ile Gly Gly Pro Met Gly Asp Ala Gly Leu Thr 250 255 260 265 GGC CGC AAG ATC ATC ATC GAC ACC TAC GGC GGC ATG GCC CGG CAT GGC 1828 Gly Arg Lys Ile Ile Ile Asp Thr Tyr Gly Gly Met Ala Arg His Gly 270 275 280 GGC GGT GCC TTC TCC GGC AAG GAC CCG TCC AAG GTG GAC CGT TCG GCG 1876 Gly Gly Ala Phe Ser Gly Lys Asp Pro Ser Lys Val Asp Arg Ser Ala 285 290 295 GCG TAC GCA ATG CGC TGG GTG GCC AAG AAC GTC GTG GCG GCG GGG CTG 1924 Ala Tyr Ala Met Arg Trp Val Ala Lys Asn Val Val Ala Ala Gly Leu 300 305 310 GCC TCG CGG TGT GAG GTG CAG GTC GCC TAC GCC ATC GGC AAG GCC GAG 1972 Ala Ser Arg Cys Glu Val Gln Val Ala Tyr Ala Ile Gly Lys Ala Glu 315 320 325 CCG GTC GGT CTG TTC GTG GAG ACC TTC GGC ACC GCC ACC GTG GAC GTG 2020 Pro Val Gly Leu Phe Val Glu Thr Phe Gly Thr Ala Thr Val Asp Val 330 335 340 345 GAG CGG ATC GAG CAG GCG ATC GGC GAG GTC TTC GAC CTC CGC CCG GCC 2068 Glu Arg Ile Glu Gln Ala Ile Gly Glu Val Phe Asp Leu Arg Pro Ala 350 355 360 GCG ATC ATC CGG GAT CTG GAC CTG CTC CGC CCG ATC TAC GCC AAG ACC 2116 Ala Ile Ile Arg Asp Leu Asp Leu Leu Arg Pro Ile Tyr Ala Lys Thr 365 370 375 GCC GCC TAC GGC CAC TTC GGC CGC GAA CTG CCC GAG TTC ACC TGG GAA 2164 Ala Ala Tyr Gly His Phe Gly Arg Glu Leu Pro Glu Phe Thr Trp Glu 380 385 390 CGC ACC GAC CGC ACC GAG CAG CTC ATC GCC GCG GCC GGC CTC TGA 2209 Arg Thr Asp Arg Thr Glu Gln Leu Ile Ala Ala Ala Gly Leu * 395 400 405 ACCGGCCGAC GTACCCCCGA GGAGTCCTCA C GTG CGC ATC GCC GTC ACC GGT 2261 Met Arg Ile Ala Val Thr Gly 1 5 TCC ATC GCC ACC GAT CAC CTC ATG GCC TTT CCC GGC CGG TTC GGG GAC 2309 Ser Ile Ala Thr Asp His Leu Met Ala Phe Pro Gly Arg Phe Gly Asp 10 15 20 CAG CTG ATC CCC GAT CAG CTC GCC CGG GTC TCG CTG TCT TTC CTC GTC 2357 Gln Leu Ile Pro Asp Gln Leu Ala Arg Val Ser Leu Ser Phe Leu Val 25 30 35 GAC GGG CTC GAG GTG CGC CGG GGC GGA GTG GCC GTC GGC ATC GCC TTC 2405 Asp Gly Leu Glu Val Arg Arg Gly Gly Val Ala Val Gly Ile Ala Phe 40 45 50 55 GGC CTG GGC CGT CCG GGC CCC ACG CCG CTG CTC GTC GGC GCC GTG GGG 2453 Gly Leu Gly Arg Pro Gly Pro Thr Pro Leu Leu Val Gly Ala Val Gly 60 65 70 AAC GAC TTC GCC GAC TAC GGG ACC TGG CCG AAG GAG CAC GGC GTC GAC 2501 Asn Asp Phe Ala Asp Tyr Gly Thr Trp Pro Lys Glu His Gly Val Asp 75 80 85 ACC GGA GGC GTT CTG GTC CCG ACC GAG CAC CAG ACC GCC CGC TTC CTG 2549 Thr Gly Gly Val Leu Val Pro Thr Glu His Gln Thr Ala Arg Phe Leu 90 95 100 TGC ATC ACC GAC CGG GAC GCC AAC CAG ATC GCG GCC TCC TAC ACG GGT 2597 Cys Ile Thr Asp Arg Asp Ala Asn Gln Ile Ala Ala Ser Tyr Thr Gly 105 110 115 GCG ATG CGG GAG GCC CGG GAC ATC GGC CTG CGG CGG ACG GGC GCT CTG 2645 Ala Met Arg Glu Ala Arg Asp Ile Gly Leu Arg Arg Thr Gly Ala Leu 120 125 130 135 CCG GCG CCC CGG CAC GGT CTG GTC CTC ATC TGC CCC GAC GAC CCG GCG 2693 Pro Ala Pro Arg His Gly Leu Val Leu Ile Cys Pro Asp Asp Pro Ala 140 145 150 GCG ATG GTG CGC CAC ACC GCG CAG TGC CGG GAG CCG GGC CTG CCG TTC 2741 Ala Met Val Arg His Thr Ala Gln Cys Arg Glu Pro Gly Leu Pro Phe 155 160 165 GTC GCC GAC CCC TCC CAG CAG CTC GCC CGG CTG GAG ACG GAC GAG GTA 2789 Val Ala Asp Pro Ser Gln Gln Leu Ala Arg Leu Glu Thr Asp Glu Val 170 175 180 CGC GCG CTG GTG CAC GGC GCC CAC TGG GTC TTC ACC AAC GAG TAC GAG 2837 Arg Ala Leu Val His Gly Ala His Trp Val Phe Thr Asn Glu Tyr Glu 185 190 195 GCC GCG CTG CTG CTC GAG CAC TCC GGC TGG AAA CAC TCC GAG ACC CTG 2885 Ala Ala Leu Leu Leu Glu His Ser Gly Trp Lys His Ser Glu Thr Leu 200 205 210 215 GAA CGG GTG GGC GCC TGG GTC ACC ACG CTC GGC GGT GCC GGG GTC CGG 2933 Glu Arg Val Gly Ala Trp Val Thr Thr Leu Gly Gly Ala Gly Val Arg 220 225 230 ATC GAG CGC GCG GGC GAG CCA CCG CTG ACG GTG CCC GCG GTC CCC GAT 2981 Ile Glu Arg Ala Gly Glu Pro Pro Leu Thr Val Pro Ala Val Pro Asp 235 240 245 GTC CCC GTG GTC GAT CCG ACC GGG ATC GGT GCC GCC TTC CGG GCC GGT 3029 Val Pro Val Val Asp Pro Thr Gly Ile Gly Ala Ala Phe Arg Ala Gly 250 255 260 TTC CTG GCC GGC GCC GGG CGC GGC CTT TCC ATC GTC TCC GCC GCC CGC 3077 Phe Leu Ala Gly Ala Gly Arg Gly Leu Ser Ile Val Ser Ala Ala Arg 265 270 275 CTG GGC TGC GTC CTG GCG GCG CGG GCG CTG GGG ACG GTC GGC CCC GCA 3125 Leu Gly Cys Val Leu Ala Ala Arg Ala Leu Gly Thr Val Gly Pro Ala 280 285 290 295 GAC CTA CCG GAC CGA TCC GGC GGA TCC GCT CGC CAC GGC GAG GGA CGC 3173 Asp Leu Pro Asp Arg Ser Gly Gly Ser Ala Arg His Gly Glu Gly Arg 300 305 310 GTA CGG CGC GGA CGC GGC GGC GCG GCT CGC CCT CGG GCT GGG CGG CCG 3221 Val Arg Arg Gly Arg Gly Gly Ala Ala Arg Pro Arg Ala Gly Arg Pro 315 320 325 CAC ATG ACC CGG CCC TGT CCC GGC TCC CGG CGC GAG CCA CCG GCC GGA 3269 His Met Thr Arg Pro Cys Pro Gly Ser Arg Arg Glu Pro Pro Ala Gly 330 335 340 CGC CCG GCA CGG GCC GCC GCC GTC ATC CGC CGC CCC GGC GCC GGC GGG 3317 Arg Pro Ala Arg Ala Ala Ala Val Ile Arg Arg Pro Gly Ala Gly Gly 345 350 355 CCG ACC GCG GGA GGC TGC CGG TGA GGACCACCCT GCGCGAGATC CTCGGCAGC 3371 Pro Thr Ala Gly Gly Cys Arg * 360 365 GCCGGCTCTC CTTCTCCCAC GAGTTCTTCC CCCCGAGGAC GGAGGCCGGC ACGCGGAC 3431 TCTGGAACGC GATCCGCCGG ATCGAACCGC TGGCCCCGAC CTTCGTCTCG GTCACCTA 3491 GCGCCGGCGG CTCCTCCCGG GACCGCACCG TCGAGGTCAC CAAGCGCATC GCCACCGA 3551 CCACCCTGCG GCCGGTCGCC CATCTCACCG CCGTCGGCCA CTCCGTCGCC GAACTGCG 3611 GCATCATCGG CCAGTACGCG GACGCCGGTG TCCGGGACGT ACTCGCCCTG CGCGGCGA 3671 CGCCGGGGGA TCCGAACGCG CCCTGGGTCC CGCATCCCGA GGGGCTCACC CACGCCCA 3731 AACTCGTCTC GCTGGTGCGC GGATCGGGCG GCTTCGGTGT GGGCGTCGCG GCCTTCCC 3791 AGCGTCATCC CCGTTCCCCC GACTGGGACA GCGAGATCCG GCACTTCGTG CGCAAGTG 3851 GGGCCGGCGC CGACTACGCC ATCACGCAGA TGTTCTTCCG GGTCGAGGAC TATCTGCG 3911 TGCGGGACCG GGTCGCGGCC GCCGGCTGCT GCACCCCGGT CATCCCCGGG ATCATGCC 3971 CCACCGACGT GCGGCAGATC GCGCGCTTCG CCGAGCTTTC CCACGCCACC TTCCCCGA 4031 GTCTCGCGCG GCGGCTGGAG GCCGCCCGCG GCAACCCGGC CGAGGGACAC CGCATCGG 4091 TCGAGTACGC CACCGCCATG GCCGGCCGGC TGCTCGCCGA AGGCGCCCCG GGACTGCA 4151 ACATCACTCT CAACCGCTCC ACCGCGACGT TGGAGATCCA CCGGAACATC CTGGGCAC 4211 CAGCCCCGGG GAGTGCCCGG CAGGTCCTCG CGGCTCCCCT CTGACCGGCG GGGACCTC 4271 GGCCCGGCCC GCACGGCGGC GGGCCCCGGC TGCGCTCCCC CGGGTCGGGT CCGGCCCG 4331 GTCCCGGCCG CCGTCCGGGC CGGCGGCGGC GGACCGTGCG CGGGCGAAGC ACACCGCC 4391 GCGTTTCCCC CGGGTCCCGG CGCCGCGGGC GGCCCCGGCG CCGGAACCCG GCCGTCCG 4451 CGCCGCGCCC CCCCGGGGGG GGACGGCCGG ACGGCCCTCT TCCGGGACGG CACTCAGC 4511 CCGCCGGAGA CGACGCCCTC GCGCAGCTCC AGATGCGAGC CGGTGAACCG GCTTCGCA 4571 CGCCGGTCGT GGGTGACCAG CACCAGCGCG CCCCCGTAGC CCGTCAGCGC CTCCTCCA 4631 TCCTCCACCA GCGCCGGTGA GAGGTGGTTG GTGGGCTCGT CCAGCAGGAG CAGACCCA 4691 GGCTCGCTGA CCAGCCGGGC CAGTTCGATG CGGCGGCGCT GACCGTACGA CAGCTCCC 4751 ACCCGCAGCC GCAGCGCCTC CGGCTCGAAC AGGCCGAGGG ACAGCAGCCG GTCGGCCT 4811 TCGTCCCGGT CGCCGGGCCG GTTGTGGGCG AAGGCCT 4848 407 amino acids amino acid linear protein 2 Met Ser Arg Arg Leu Phe Thr Ser Glu Ser Val Thr Glu Gly His Pro 1 5 10 15 Asp Lys Ile Ala Asp Arg Ile Ser Asp Thr Val Leu Asp Ala Leu Leu 20 25 30 Ala Arg Asp Pro Arg Ala Arg Val Ala Val Glu Thr Leu Ile Thr Thr 35 40 45 Gly Gln Val His Ile Ala Gly Glu Val Thr Thr Thr Ala Tyr Ala Pro 50 55 60 Ile Ala Gln Leu Val Arg Asp Thr Val Leu Ser Ile Gly Tyr Asp Ser 65 70 75 80 Ser Ala Lys Gly Phe Asp Gly Ala Ser Cys Gly Val Ser Val Ser Ile 85 90 95 Gly Ala Gln Ser Pro Asp Ile Ala Arg Gly Val Asp Thr Ala Tyr Glu 100 105 110 Arg Arg Gly Gly Gly Thr Ala Pro Gly Gly Pro Gly Asp Glu Leu Asp 115 120 125 Arg Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Cys Asp 130 135 140 Glu Thr Pro Glu Leu Met Pro Leu Pro Ile Asn Leu Ala His Arg Leu 145 150 155 160 Ser Arg Arg Leu Ser Glu Val Arg Lys Asn Gly Thr Ile Pro Tyr Leu 165 170 175 Arg Pro Asp Gly Lys Thr Gln Val Thr Ile Glu Tyr Asp Gly Asp Lys 180 185 190 Ala Val Arg Leu Asp Thr Val Val Val Ser Ser Gln His Ala Ser Gly 195 200 205 Ile Asp Leu Asp Ser Leu Leu Ala Pro Asp Ile Arg Arg His Val Val 210 215 220 Glu Pro Val Leu Ala Gly Leu Ala Glu Asp Gly Ile Lys Leu Asp Thr 225 230 235 240 Ala Gly Tyr Arg Leu Leu Val Asn Pro Thr Gly Arg Phe Glu Ile Gly 245 250 255 Gly Pro Met Gly Asp Ala Gly Leu Thr Gly Arg Lys Ile Ile Ile Asp 260 265 270 Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys 275 280 285 Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr Ala Met Arg Trp Val 290 295 300 Ala Lys Asn Val Val Ala Ala Gly Leu Ala Ser Arg Cys Glu Val Gln 305 310 315 320 Val Ala Tyr Ala Ile Gly Lys Ala Glu Pro Val Gly Leu Phe Val Glu 325 330 335 Thr Phe Gly Thr Ala Thr Val Asp Val Glu Arg Ile Glu Gln Ala Ile 340 345 350 Gly Glu Val Phe Asp Leu Arg Pro Ala Ala Ile Ile Arg Asp Leu Asp 355 360 365 Leu Leu Arg Pro Ile Tyr Ala Lys Thr Ala Ala Tyr Gly His Phe Gly 370 375 380 Arg Glu Leu Pro Glu Phe Thr Trp Glu Arg Thr Asp Arg Thr Glu Gln 385 390 395 400 Leu Ile Ala Ala Ala Gly Leu 405 366 amino acids amino acid linear protein 3 Met Arg Ile Ala Val Thr Gly Ser Ile Ala Thr Asp His Leu Met Ala 1 5 10 15 Phe Pro Gly Arg Phe Gly Asp Gln Leu Ile Pro Asp Gln Leu Ala Arg 20 25 30 Val Ser Leu Ser Phe Leu Val Asp Gly Leu Glu Val Arg Arg Gly Gly 35 40 45 Val Ala Val Gly Ile Ala Phe Gly Leu Gly Arg Pro Gly Pro Thr Pro 50 55 60 Leu Leu Val Gly Ala Val Gly Asn Asp Phe Ala Asp Tyr Gly Thr Trp 65 70 75 80 Pro Lys Glu His Gly Val Asp Thr Gly Gly Val Leu Val Pro Thr Glu 85 90 95 His Gln Thr Ala Arg Phe Leu Cys Ile Thr Asp Arg Asp Ala Asn Gln 100 105 110 Ile Ala Ala Ser Tyr Thr Gly Ala Met Arg Glu Ala Arg Asp Ile Gly 115 120 125 Leu Arg Arg Thr Gly Ala Leu Pro Ala Pro Arg His Gly Leu Val Leu 130 135 140 Ile Cys Pro Asp Asp Pro Ala Ala Met Val Arg His Thr Ala Gln Cys 145 150 155 160 Arg Glu Pro Gly Leu Pro Phe Val Ala Asp Pro Ser Gln Gln Leu Ala 165 170 175 Arg Leu Glu Thr Asp Glu Val Arg Ala Leu Val His Gly Ala His Trp 180 185 190 Val Phe Thr Asn Glu Tyr Glu Ala Ala Leu Leu Leu Glu His Ser Gly 195 200 205 Trp Lys His Ser Glu Thr Leu Glu Arg Val Gly Ala Trp Val Thr Thr 210 215 220 Leu Gly Gly Ala Gly Val Arg Ile Glu Arg Ala Gly Glu Pro Pro Leu 225 230 235 240 Thr Val Pro Ala Val Pro Asp Val Pro Val Val Asp Pro Thr Gly Ile 245 250 255 Gly Ala Ala Phe Arg Ala Gly Phe Leu Ala Gly Ala Gly Arg Gly Leu 260 265 270 Ser Ile Val Ser Ala Ala Arg Leu Gly Cys Val Leu Ala Ala Arg Ala 275 280 285 Leu Gly Thr Val Gly Pro Ala Asp Leu Pro Asp Arg Ser Gly Gly Ser 290 295 300 Ala Arg His Gly Glu Gly Arg Val Arg Arg Gly Arg Gly Gly Ala Ala 305 310 315 320 Arg Pro Arg Ala Gly Arg Pro His Met Thr Arg Pro Cys Pro Gly Ser 325 330 335 Arg Arg Glu Pro Pro Ala Gly Arg Pro Ala Arg Ala Ala Ala Val Ile 340 345 350 Arg Arg Pro Gly Ala Gly Gly Pro Thr Ala Gly Gly Cys Arg 355 360 365 4848 base pairs nucleic acid single linear DNA (genomic) NO NO CDS 3338..4255 4 TCATGACACC TCTGGTGAGG GGGGGCAGCT CAGCTCACCC CCGATCCGGA CACGCCGGGT 60 CCGCGGGGAG TCCCGCGGCA CCGCCGCTCG TCAACGCAGC GGGGCGAGCA CATGTCTTT 120 TCACATTTCT CCTTGGGGGG GCGAGGCCGG CGCCCGGCCG TCGCCTTTCG TGAATGCGG 180 GGAGCCGCAT CGCGAACGCG ATGCGGCTCC GGGGAGACTT CTCGAACCCG AGAAGGAAG 240 GGATTCGGCG ATACGGTTCC GGAATACCGC GGCTCGTCAC GAAGCCGGTT CCGGATCAG 300 GGTGCGGCCG TTCCAGGGCA CACCACGCCT GTTCCCCGGA GCGTGACAGG ACGCTCATC 360 GGGTGTCAAC GCACCATTCG ACGGAGGGAG TTGGGCCGCA CCGGCCGGAG GGGTCCGAC 420 AGGGGTTTCG GGCGGGATCG AGAAACACTC GAGAAGCGGC GGAAAACACC GTGCGGCTG 480 CCGGACCCAG GCGCCACCCG TTCTTCATCG GCTCTCCAGC CGGCCTTGAG CGCCCCGGC 540 GGCCACCACC CGCCGAAAAC CGGGACCAAG GATTCACCGG GTTCGTGACC GTATTGCGG 600 GTGGGATCCC GGGGATGGGA TGCCGGATGG ATTTCTGAGC AGGCGTCGCC CGGCAGCCG 660 CGGGCCGTGG CCGGAACCGG CCGGGCCGGT CGGCGGGGTG TGCGCGGTGC CGGCCGAAG 720 CGTGGGCCGG AGCGCGGACC CGAGCGCCGT GTCAGCGCCG TGTCAGGGCT TTGGGGGCG 780 GCCCTCACAT ACTCGACCCG CGTTCACGGA GAGGAAGGCA GGGAAGGGCC CGTCCGCAC 840 GCGACCGGTG CCGGCGCCCC CGCCCGATGC CCGGCGCCGC CCCACCGAGC CGGACGCAC 900 GGAGGTTCCC ACCGCCCGGC CGGACGCCGG CGTGCCGTTC AGCCGGTGCG CGGGCCGGC 960 CGAGATCTCA CACCACTGGA GAGCCATGTC ACGTCGCCTG TTCACCTCGG AGTCCGTG 1020 CGAGGGCCAC CCCGACAAGA TCGCCGACCG GATCAGCGAC ACCGTCCTCG ATGCCCTG 1080 CGCCCGGGAC CCGCGGGCCA GGGTCGCCGT CGAGACCCTG ATCACCACCG GTCAGGTC 1140 TATCGCCGGC GAGGTCACCA CCACCGCGTA CGCGCCCATC GCCCAACTGG TGCGCGAC 1200 CGTCCTGTCC ATCGGCTACG ACTCCTCGGC CAAGGGCTTC GACGGCGCCT CGTGCGGG 1260 GTCGGTCTCC ATCGGCGCGC AGTCCCCGGA CATCGCCCGG GGTGTGGACA CCGCGTAC 1320 GCGGCGGGGC GGGGGCACGG CCCCGGGCGG ACCGGGTGAC GAGCTGGACC GGCAGGGC 1380 GGGCGACCAG GGCCTGATGT TCGGCTACGC CTGCGACGAG ACCCCCGAGC TGATGCCG 1440 GCCGATCAAC CTGGCGCACC GGCTCTCCCG GCGGCTGTCG GAGGTGCGGA AGAACGGC 1500 GATCCCCTAC CTCCGCCCCG ACGGCAAGAC CCAGGTCACC ATCGAGTACG ACGGCGAC 1560 GGCGGTCCGC CTCGACACGG TCGTGGTCTC CTCCCAGCAC GCCTCCGGCA TCGACCTG 1620 CTCGCTACTG GCGCCCGACA TCCGCCGGCA TGTCGTGGAG CCCGTCCTCG CCGGACTG 1680 CGAGGACGGC ATCAAGCTGG ACACCGCGGG CTACCGGCTG CTGGTCAATC CCACCGGC 1740 GTTCGAGATC GGCGGCCCGA TGGGCGACGC GGGCCTGACC GGCCGCAAGA TCATCATC 1800 CACCTACGGC GGCATGGCCC GGCATGGCGG CGGTGCCTTC TCCGGCAAGG ACCCGTCC 1860 GGTGGACCGT TCGGCGGCGT ACGCAATGCG CTGGGTGGCC AAGAACGTCG TGGCGGCG 1920 GCTGGCCTCG CGGTGTGAGG TGCAGGTCGC CTACGCCATC GGCAAGGCCG AGCCGGTC 1980 TCTGTTCGTG GAGACCTTCG GCACCGCCAC CGTGGACGTG GAGCGGATCG AGCAGGCG 2040 CGGCGAGGTC TTCGACCTCC GCCCGGCCGC GATCATCCGG GATCTGGACC TGCTCCGC 2100 GATCTACGCC AAGACCGCCG CCTACGGCCA CTTCGGCCGC GAACTGCCCG AGTTCACC 2160 GGAACGCACC GACCGCACCG AGCAGCTCAT CGCCGCGGCC GGCCTCTGAA CCGGCCGA 2220 TACCCCCGAG GAGTCCTCAC GTGCGCATCG CCGTCACCGG TTCCATCGCC ACCGATCA 2280 TCATGGCCTT TCCCGGCCGG TTCGGGGACC AGCTGATCCC CGATCAGCTC GCCCGGGT 2340 CGCTGTCTTT CCTCGTCGAC GGGCTCGAGG TGCGCCGGGG CGGAGTGGCC GTCGGCAT 2400 CCTTCGGCCT GGGCCGTCCG GGCCCCACGC CGCTGCTCGT CGGCGCCGTG GGGAACGA 2460 TCGCCGACTA CGGGACCTGG CCGAAGGAGC ACGGCGTCGA CACCGGAGGC GTTCTGGT 2520 CGACCGAGCA CCAGACCGCC CGCTTCCTGT GCATCACCGA CCGGGACGCC AACCAGAT 2580 CGGCCTCCTA CACGGGTGCG ATGCGGGAGG CCCGGGACAT CGGCCTGCGG CGGACGGG 2640 CTCTGCCGGC GCCCCGGCAC GGTCTGGTCC TCATCTGCCC CGACGACCCG GCGGCGAT 2700 TGCGCCACAC CGCGCAGTGC CGGGAGCCGG GCCTGCCGTT CGTCGCCGAC CCCTCCCA 2760 AGCTCGCCCG GCTGGAGACG GACGAGGTAC GCGCGCTGGT GCACGGCGCC CACTGGGT 2820 TCACCAACGA GTACGAGGCC GCGCTGCTGC TCGAGCACTC CGGCTGGAAA CACTCCGA 2880 CCCTGGAACG GGTGGGCGCC TGGGTCACCA CGCTCGGCGG TGCCGGGGTC CGGATCGA 2940 GCGCGGGCGA GCCACCGCTG ACGGTGCCCG CGGTCCCCGA TGTCCCCGTG GTCGATCC 3000 CCGGGATCGG TGCCGCCTTC CGGGCCGGTT TCCTGGCCGG CGCCGGGCGC GGCCTTTC 3060 TCGTCTCCGC CGCCCGCCTG GGCTGCGTCC TGGCGGCGCG GGCGCTGGGG ACGGTCGG 3120 CCGCAGACCT ACCGGACCGA TCCGGCGGAT CCGCTCGCCA CGGCGAGGGA CGCGTACG 3180 GCGGACGCGG CGGCGCGGCT CGCCCTCGGG CTGGGCGGCC GCACATGACC CGGCCCTG 3240 CCGGCTCCCG GCGCGAGCCA CCGGCCGGAC GCCCGGCACG GGCCGCCGCC GTCATCCG 3300 GCCCCGGCGC CGGCGGGCCG ACCGCGGGAG GCTGCCG GTG AGG ACC ACC CTG CG 3355 Met Arg Thr Thr Leu Arg 1 5 GAG ATC CTC GGC AGC GGC CGG CTC TCC TTC TCC CAC GAG TTC TTC CCC 3403 Glu Ile Leu Gly Ser Gly Arg Leu Ser Phe Ser His Glu Phe Phe Pro 10 15 20 CCG AGG ACG GAG GCC GGC ACG CGG ACG CTC TGG AAC GCG ATC CGC CGG 3451 Pro Arg Thr Glu Ala Gly Thr Arg Thr Leu Trp Asn Ala Ile Arg Arg 25 30 35 ATC GAA CCG CTG GCC CCG ACC TTC GTC TCG GTC ACC TAC GGC GCC GGC 3499 Ile Glu Pro Leu Ala Pro Thr Phe Val Ser Val Thr Tyr Gly Ala Gly 40 45 50 GGC TCC TCC CGG GAC CGC ACC GTC GAG GTC ACC AAG CGC ATC GCC ACC 3547 Gly Ser Ser Arg Asp Arg Thr Val Glu Val Thr Lys Arg Ile Ala Thr 55 60 65 70 GAC ACC ACC CTG CGG CCG GTC GCC CAT CTC ACC GCC GTC GGC CAC TCC 3595 Asp Thr Thr Leu Arg Pro Val Ala His Leu Thr Ala Val Gly His Ser 75 80 85 GTC GCC GAA CTG CGC CGC ATC ATC GGC CAG TAC GCG GAC GCC GGT GTC 3643 Val Ala Glu Leu Arg Arg Ile Ile Gly Gln Tyr Ala Asp Ala Gly Val 90 95 100 CGG GAC GTA CTC GCC CTG CGC GGC GAC CCG CCG GGG GAT CCG AAC GCG 3691 Arg Asp Val Leu Ala Leu Arg Gly Asp Pro Pro Gly Asp Pro Asn Ala 105 110 115 CCC TGG GTC CCG CAT CCC GAG GGG CTC ACC CAC GCC CAT GAA CTC GTC 3739 Pro Trp Val Pro His Pro Glu Gly Leu Thr His Ala His Glu Leu Val 120 125 130 TCG CTG GTG CGC GGA TCG GGC GGC TTC GGT GTG GGC GTC GCG GCC TTC 3787 Ser Leu Val Arg Gly Ser Gly Gly Phe Gly Val Gly Val Ala Ala Phe 135 140 145 150 CCC GAG CGT CAT CCC CGT TCC CCC GAC TGG GAC AGC GAG ATC CGG CAC 3835 Pro Glu Arg His Pro Arg Ser Pro Asp Trp Asp Ser Glu Ile Arg His 155 160 165 TTC GTG CGC AAG TGC CGG GCC GGC GCC GAC TAC GCC ATC ACG CAG ATG 3883 Phe Val Arg Lys Cys Arg Ala Gly Ala Asp Tyr Ala Ile Thr Gln Met 170 175 180 TTC TTC CGG GTC GAG GAC TAT CTG CGG CTG CGG GAC CGG GTC GCG GCC 3931 Phe Phe Arg Val Glu Asp Tyr Leu Arg Leu Arg Asp Arg Val Ala Ala 185 190 195 GCC GGC TGC TGC ACC CCG GTC ATC CCC GGG ATC ATG CCC GCC ACC GAC 3979 Ala Gly Cys Cys Thr Pro Val Ile Pro Gly Ile Met Pro Ala Thr Asp 200 205 210 GTG CGG CAG ATC GCG CGC TTC GCC GAG CTT TCC CAC GCC ACC TTC CCC 4027 Val Arg Gln Ile Ala Arg Phe Ala Glu Leu Ser His Ala Thr Phe Pro 215 220 225 230 GAA GGT CTC GCG CGG CGG CTG GAG GCC GCC CGC GGC AAC CCG GCC GAG 4075 Glu Gly Leu Ala Arg Arg Leu Glu Ala Ala Arg Gly Asn Pro Ala Glu 235 240 245 GGA CAC CGC ATC GGG GTC GAG TAC GCC ACC GCC ATG GCC GGC CGG CTG 4123 Gly His Arg Ile Gly Val Glu Tyr Ala Thr Ala Met Ala Gly Arg Leu 250 255 260 CTC GCC GAA GGC GCC CCG GGA CTG CAC TAC ATC ACT CTC AAC CGC TCC 4171 Leu Ala Glu Gly Ala Pro Gly Leu His Tyr Ile Thr Leu Asn Arg Ser 265 270 275 ACC GCG ACG TTG GAG ATC CAC CGG AAC ATC CTG GGC ACA CCA GCC CCG 4219 Thr Ala Thr Leu Glu Ile His Arg Asn Ile Leu Gly Thr Pro Ala Pro 280 285 290 GGG AGT GCC CGG CAG GTC CTC GCG GCT CCC CTC TGA CCGGCGGGGA 4265 Gly Ser Ala Arg Gln Val Leu Ala Ala Pro Leu * 295 300 305 CCTCTCGGCC CGGCCCGCAC GGCGGCGGGC CCCGGCTGCG CTCCCCCGGG TCGGGTCC 4325 CCCGCCGTCC CGGCCGCCGT CCGGGCCGGC GGCGGCGGAC CGTGCGCGGG CGAAGCAC 4385 CGCCGCGCGT TTCCCCCGGG TCCCGGCGCC GCGGGCGGCC CCGGCGCCGG AACCCGGC 4445 TCCGCTCGCC GCGCCCCCCC GGGGGGGGAC GGCCGGACGG CCCTCTTCCG GGACGGCA 4505 CAGCGTCCGC CGGAGACGAC GCCCTCGCGC AGCTCCAGAT GCGAGCCGGT GAACCGGC 4565 CGCATCCGCC GGTCGTGGGT GACCAGCACC AGCGCGCCCC CGTAGCCCGT CAGCGCCT 4625 TCCAACTCCT CCACCAGCGC CGGTGAGAGG TGGTTGGTGG GCTCGTCCAG CAGGAGCA 4685 CCCACCGGCT CGCTGACCAG CCGGGCCAGT TCGATGCGGC GGCGCTGACC GTACGACA 4745 TCCCCGACCC GCAGCCGCAG CGCCTCCGGC TCGAACAGGC CGAGGGACAG CAGCCGGT 4805 GCCTGTTCGT CCCGGTCGCC GGGCCGGTTG TGGGCGAAGG CCT 4848 305 amino acids amino acid linear protein 5 Met Arg Thr Thr Leu Arg Glu Ile Leu Gly Ser Gly Arg Leu Ser Phe 1 5 10 15 Ser His Glu Phe Phe Pro Pro Arg Thr Glu Ala Gly Thr Arg Thr Leu 20 25 30 Trp Asn Ala Ile Arg Arg Ile Glu Pro Leu Ala Pro Thr Phe Val Ser 35 40 45 Val Thr Tyr Gly Ala Gly Gly Ser Ser Arg Asp Arg Thr Val Glu Val 50 55 60 Thr Lys Arg Ile Ala Thr Asp Thr Thr Leu Arg Pro Val Ala His Leu 65 70 75 80 Thr Ala Val Gly His Ser Val Ala Glu Leu Arg Arg Ile Ile Gly Gln 85 90 95 Tyr Ala Asp Ala Gly Val Arg Asp Val Leu Ala Leu Arg Gly Asp Pro 100 105 110 Pro Gly Asp Pro Asn Ala Pro Trp Val Pro His Pro Glu Gly Leu Thr 115 120 125 His Ala His Glu Leu Val Ser Leu Val Arg Gly Ser Gly Gly Phe Gly 130 135 140 Val Gly Val Ala Ala Phe Pro Glu Arg His Pro Arg Ser Pro Asp Trp 145 150 155 160 Asp Ser Glu Ile Arg His Phe Val Arg Lys Cys Arg Ala Gly Ala Asp 165 170 175 Tyr Ala Ile Thr Gln Met Phe Phe Arg Val Glu Asp Tyr Leu Arg Leu 180 185 190 Arg Asp Arg Val Ala Ala Ala Gly Cys Cys Thr Pro Val Ile Pro Gly 195 200 205 Ile Met Pro Ala Thr Asp Val Arg Gln Ile Ala Arg Phe Ala Glu Leu 210 215 220 Ser His Ala Thr Phe Pro Glu Gly Leu Ala Arg Arg Leu Glu Ala Ala 225 230 235 240 Arg Gly Asn Pro Ala Glu Gly His Arg Ile Gly Val Glu Tyr Ala Thr 245 250 255 Ala Met Ala Gly Arg Leu Leu Ala Glu Gly Ala Pro Gly Leu His Tyr 260 265 270 Ile Thr Leu Asn Arg Ser Thr Ala Thr Leu Glu Ile His Arg Asn Ile 275 280 285 Leu Gly Thr Pro Ala Pro Gly Ser Ala Arg Gln Val Leu Ala Ala Pro 290 295 300 Leu 305 4848 base pairs nucleic acid single linear mRNA NO NO 6 UCAUGACACC UCUGGUGAGG GGGGGCAGCU CAGCUCACCC CCGAUCCGGA CACGCCGGGU 60 CCGCGGGGAG UCCCGCGGCA CCGCCGCUCG UCAACGCAGC GGGGCGAGCA CAUGUCUUU 120 UCACAUUUCU CCUUGGGGGG GCGAGGCCGG CGCCCGGCCG UCGCCUUUCG UGAAUGCGG 180 GGAGCCGCAU CGCGAACGCG AUGCGGCUCC GGGGAGACUU CUCGAACCCG AGAAGGAAG 240 GGAUUCGGCG AUACGGUUCC GGAAUACCGC GGCUCGUCAC GAAGCCGGUU CCGGAUCAG 300 GGUGCGGCCG UUCCAGGGCA CACCACGCCU GUUCCCCGGA GCGUGACAGG ACGCUCAUC 360 GGGUGUCAAC GCACCAUUCG ACGGAGGGAG UUGGGCCGCA CCGGCCGGAG GGGUCCGAC 420 AGGGGUUUCG GGCGGGAUCG AGAAACACUC GAGAAGCGGC GGAAAACACC GUGCGGCUG 480 CCGGACCCAG GCGCCACCCG UUCUUCAUCG GCUCUCCAGC CGGCCUUGAG CGCCCCGGC 540 GGCCACCACC CGCCGAAAAC CGGGACCAAG GAUUCACCGG GUUCGUGACC GUAUUGCGG 600 GUGGGAUCCC GGGGAUGGGA UGCCGGAUGG AUUUCUGAGC AGGCGUCGCC CGGCAGCCG 660 CGGGCCGUGG CCGGAACCGG CCGGGCCGGU CGGCGGGGUG UGCGCGGUGC CGGCCGAAG 720 CGUGGGCCGG AGCGCGGACC CGAGCGCCGU GUCAGCGCCG UGUCAGGGCU UUGGGGGCG 780 GCCCUCACAU ACUCGACCCG CGUUCACGGA GAGGAAGGCA GGGAAGGGCC CGUCCGCAC 840 GCGACCGGUG CCGGCGCCCC CGCCCGAUGC CCGGCGCCGC CCCACCGAGC CGGACGCAC 900 GGAGGUUCCC ACCGCCCGGC CGGACGCCGG CGUGCCGUUC AGCCGGUGCG CGGGCCGGC 960 CGAGAUCUCA CACCACUGGA GAGCCAUGUC ACGUCGCCUG UUCACCUCGG AGUCCGUG 1020 CGAGGGCCAC CCCGACAAGA UCGCCGACCG GAUCAGCGAC ACCGUCCUCG AUGCCCUG 1080 CGCCCGGGAC CCGCGGGCCA GGGUCGCCGU CGAGACCCUG AUCACCACCG GUCAGGUC 1140 UAUCGCCGGC GAGGUCACCA CCACCGCGUA CGCGCCCAUC GCCCAACUGG UGCGCGAC 1200 CGUCCUGUCC AUCGGCUACG ACUCCUCGGC CAAGGGCUUC GACGGCGCCU CGUGCGGG 1260 GUCGGUCUCC AUCGGCGCGC AGUCCCCGGA CAUCGCCCGG GGUGUGGACA CCGCGUAC 1320 GCGGCGGGGC GGGGGCACGG CCCCGGGCGG ACCGGGUGAC GAGCUGGACC GGCAGGGC 1380 GGGCGACCAG GGCCUGAUGU UCGGCUACGC CUGCGACGAG ACCCCCGAGC UGAUGCCG 1440 GCCGAUCAAC CUGGCGCACC GGCUCUCCCG GCGGCUGUCG GAGGUGCGGA AGAACGGC 1500 GAUCCCCUAC CUCCGCCCCG ACGGCAAGAC CCAGGUCACC AUCGAGUACG ACGGCGAC 1560 GGCGGUCCGC CUCGACACGG UCGUGGUCUC CUCCCAGCAC GCCUCCGGCA UCGACCUG 1620 CUCGCUACUG GCGCCCGACA UCCGCCGGCA UGUCGUGGAG CCCGUCCUCG CCGGACUG 1680 CGAGGACGGC AUCAAGCUGG ACACCGCGGG CUACCGGCUG CUGGUCAAUC CCACCGGC 1740 GUUCGAGAUC GGCGGCCCGA UGGGCGACGC GGGCCUGACC GGCCGCAAGA UCAUCAUC 1800 CACCUACGGC GGCAUGGCCC GGCAUGGCGG CGGUGCCUUC UCCGGCAAGG ACCCGUCC 1860 GGUGGACCGU UCGGCGGCGU ACGCAAUGCG CUGGGUGGCC AAGAACGUCG UGGCGGCG 1920 GCUGGCCUCG CGGUGUGAGG UGCAGGUCGC CUACGCCAUC GGCAAGGCCG AGCCGGUC 1980 UCUGUUCGUG GAGACCUUCG GCACCGCCAC CGUGGACGUG GAGCGGAUCG AGCAGGCG 2040 CGGCGAGGUC UUCGACCUCC GCCCGGCCGC GAUCAUCCGG GAUCUGGACC UGCUCCGC 2100 GAUCUACGCC AAGACCGCCG CCUACGGCCA CUUCGGCCGC GAACUGCCCG AGUUCACC 2160 GGAACGCACC GACCGCACCG AGCAGCUCAU CGCCGCGGCC GGCCUCUGAA CCGGCCGA 2220 UACCCCCGAG GAGUCCUCAC GUGCGCAUCG CCGUCACCGG UUCCAUCGCC ACCGAUCA 2280 UCAUGGCCUU UCCCGGCCGG UUCGGGGACC AGCUGAUCCC CGAUCAGCUC GCCCGGGU 2340 CGCUGUCUUU CCUCGUCGAC GGGCUCGAGG UGCGCCGGGG CGGAGUGGCC GUCGGCAU 2400 CCUUCGGCCU GGGCCGUCCG GGCCCCACGC CGCUGCUCGU CGGCGCCGUG GGGAACGA 2460 UCGCCGACUA CGGGACCUGG CCGAAGGAGC ACGGCGUCGA CACCGGAGGC GUUCUGGU 2520 CGACCGAGCA CCAGACCGCC CGCUUCCUGU GCAUCACCGA CCGGGACGCC AACCAGAU 2580 CGGCCUCCUA CACGGGUGCG AUGCGGGAGG CCCGGGACAU CGGCCUGCGG CGGACGGG 2640 CUCUGCCGGC GCCCCGGCAC GGUCUGGUCC UCAUCUGCCC CGACGACCCG GCGGCGAU 2700 UGCGCCACAC CGCGCAGUGC CGGGAGCCGG GCCUGCCGUU CGUCGCCGAC CCCUCCCA 2760 AGCUCGCCCG GCUGGAGACG GACGAGGUAC GCGCGCUGGU GCACGGCGCC CACUGGGU 2820 UCACCAACGA GUACGAGGCC GCGCUGCUGC UCGAGCACUC CGGCUGGAAA CACUCCGA 2880 CCCUGGAACG GGUGGGCGCC UGGGUCACCA CGCUCGGCGG UGCCGGGGUC CGGAUCGA 2940 GCGCGGGCGA GCCACCGCUG ACGGUGCCCG CGGUCCCCGA UGUCCCCGUG GUCGAUCC 3000 CCGGGAUCGG UGCCGCCUUC CGGGCCGGUU UCCUGGCCGG CGCCGGGCGC GGCCUUUC 3060 UCGUCUCCGC CGCCCGCCUG GGCUGCGUCC UGGCGGCGCG GGCGCUGGGG ACGGUCGG 3120 CCGCAGACCU ACCGGACCGA UCCGGCGGAU CCGCUCGCCA CGGCGAGGGA CGCGUACG 3180 GCGGACGCGG CGGCGCGGCU CGCCCUCGGG CUGGGCGGCC GCACAUGACC CGGCCCUG 3240 CCGGCUCCCG GCGCGAGCCA CCGGCCGGAC GCCCGGCACG GGCCGCCGCC GUCAUCCG 3300 GCCCCGGCGC CGGCGGGCCG ACCGCGGGAG GCUGCCGGUG AGGACCACCC UGCGCGAG 3360 CCUCGGCAGC GGCCGGCUCU CCUUCUCCCA CGAGUUCUUC CCCCCGAGGA CGGAGGCC 3420 CACGCGGACG CUCUGGAACG CGAUCCGCCG GAUCGAACCG CUGGCCCCGA CCUUCGUC 3480 GGUCACCUAC GGCGCCGGCG GCUCCUCCCG GGACCGCACC GUCGAGGUCA CCAAGCGC 3540 CGCCACCGAC ACCACCCUGC GGCCGGUCGC CCAUCUCACC GCCGUCGGCC ACUCCGUC 3600 CGAACUGCGC CGCAUCAUCG GCCAGUACGC GGACGCCGGU GUCCGGGACG UACUCGCC 3660 GCGCGGCGAC CCGCCGGGGG AUCCGAACGC GCCCUGGGUC CCGCAUCCCG AGGGGCUC 3720 CCACGCCCAU GAACUCGUCU CGCUGGUGCG CGGAUCGGGC GGCUUCGGUG UGGGCGUC 3780 GGCCUUCCCC GAGCGUCAUC CCCGUUCCCC CGACUGGGAC AGCGAGAUCC GGCACUUC 3840 GCGCAAGUGC CGGGCCGGCG CCGACUACGC CAUCACGCAG AUGUUCUUCC GGGUCGAG 3900 CUAUCUGCGG CUGCGGGACC GGGUCGCGGC CGCCGGCUGC UGCACCCCGG UCAUCCCC 3960 GAUCAUGCCC GCCACCGACG UGCGGCAGAU CGCGCGCUUC GCCGAGCUUU CCCACGCC 4020 CUUCCCCGAA GGUCUCGCGC GGCGGCUGGA GGCCGCCCGC GGCAACCCGG CCGAGGGA 4080 CCGCAUCGGG GUCGAGUACG CCACCGCCAU GGCCGGCCGG CUGCUCGCCG AAGGCGCC 4140 GGGACUGCAC UACAUCACUC UCAACCGCUC CACCGCGACG UUGGAGAUCC ACCGGAAC 4200 CCUGGGCACA CCAGCCCCGG GGAGUGCCCG GCAGGUCCUC GCGGCUCCCC UCUGACCG 4260 GGGGACCUCU CGGCCCGGCC CGCACGGCGG CGGGCCCCGG CUGCGCUCCC CCGGGUCG 4320 UCCGGCCCGC CGUCCCGGCC GCCGUCCGGG CCGGCGGCGG CGGACCGUGC GCGGGCGA 4380 CACACCGCCG CGCGUUUCCC CCGGGUCCCG GCGCCGCGGG CGGCCCCGGC GCCGGAAC 4440 GGCCGUCCGC UCGCCGCGCC CCCCCGGGGG GGGACGGCCG GACGGCCCUC UUCCGGGA 4500 GCACUCAGCG UCCGCCGGAG ACGACGCCCU CGCGCAGCUC CAGAUGCGAG CCGGUGAA 4560 GGCUUCGCAU CCGCCGGUCG UGGGUGACCA GCACCAGCGC GCCCCCGUAG CCCGUCAG 4620 CCUCCUCCAA CUCCUCCACC AGCGCCGGUG AGAGGUGGUU GGUGGGCUCG UCCAGCAG 4680 GCAGACCCAC CGGCUCGCUG ACCAGCCGGG CCAGUUCGAU GCGGCGGCGC UGACCGUA 4740 ACAGCUCCCC GACCCGCAGC CGCAGCGCCU CCGGCUCGAA CAGGCCGAGG GACAGCAG 4800 GGUCGGCCUG UUCGUCCCGG UCGCCGGGCC GGUUGUGGGC GAAGGCCU 4848 

We claim;
 1. A substantially pure SAM symthetase from Streptomyces fradiae having the amino acid sequence; Met Ser Arg Arg Leu Phe Thr Ser Glu Ser Val Thr Glu Gly His Pro   1               5                  10                 15 Asp Lys Ile Ala Asp Arg Ile Ser Asp Thr Val Leu Asp Ala Leu Leu              20                  25                  30 Ala Arg Asp Pro Arg Ala Arg Val Ala Val Glu Thr Leu Ile Thr Thr          35                  40                  45 Gly Gln Val His Ile Ala Gly Glu Val Thr Thr Thr Ala Tyr Ala Pro      50                  55                  60 Ile Ala Gln Leu Val Arg Asp Thr Val Leu Ser Ile Gly Tyr Asp Ser  65                  70                  75                  80 Ser Ala Lys Gly Phe Asp Gly Ala Ser Cys Gly Val Ser Val Ser Ile                  85                  90                  95 Gly Ala Gln Ser Pro Asp Ile Ala Arg Gly Val Asp Thr Ala Tyr Glu             100                 105                 110 Arg Arg Gly Gly Gly Thr Ala Pro Gly Gly Pro Gly Asp Glu Leu Asp         115                 120                 125 Arg Gln Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Cys Asp     130                 135                 140 Glu Thr Pro Glu Leu Met Pro Leu Pro Ile Asn Leu Ala His Arg Leu 145                 150                 155                 160 Ser Arg Arg Leu Ser Glu Val Arg Lys Asn Gly Thr Ile Pro Tyr Leu                 165                 170                 175 Arg Pro Asp Gly Lys Thr Gln Val Thr Ile Glu Tyr Asp Gly Asp Lys             180                 185                 190 Ala Val Arg Leu Asp Thr Val Val Val Ser Ser Gln His Ala Ser Gly         195                 200                 205 Ile Asp Leu Asp Ser Leu Leu Ala Pro Asp Ile Arg Arg His Val Val     210                 215                 220 Glu Pro Val Leu Ala Gly Leu Ala Glu Asp Gly Ile Lys Leu Asp Thr 225                 230                 235                 240 Ala Gly Tyr Arg Leu Leu Val Asn Pro Thr Gly Arg Phe Glu Ile Gly                 245                 250                 255 Gly Pro Met Gly Asp Ala Gly Leu Thr Gly Arg Lys Ile Ile Ile Asp             260                 265                 270 Thr Tyr Gly Gly Met Ala Arg His Gly Gly Gly Ala Phe Ser Gly Lys         275                 280                 285 Asp Pro Ser Lys Val Asp Arg Ser Ala Ala Tyr Ala Met Arg Trp Val     290                 295                 300 Ala Lys Asn Val Val Ala Ala Gly Leu Ala Ser Arg Cys Glu Val Gln 305                 310                 315                 320 Val Ala Tyr Ala Ile Gly Lys Ala Glu Pro Val Gly Leu Phe Val Glu                 325                 330                 335 Thr Phe Gly Thr Ala Thr Val Asp Val Glu Arg Ile Glu Gln Ala Ile             340                 345                 350 Gly Glu Val Phe Asp Leu Arg Pro Ala Ala Ile Ile Arg Asp Leu Asp         355                 360                 365 Leu Leu Arg Pro Ile Tyr Ala Lys Thr Ala Ala Tyr Gly His Phe Gly     370                 375                 380 Arg Glu Leu Pro Glu Phe Thr Trp Glu Arg Thr Asp Arg Thr Glu Gln 385                 390                 395                 400 Leu Ile Ala Ala Ala Gly Leu  *                 405

which is SEQ ID NO
 2. 2. A substantially pure methyltransferase from Streptomyces fradiae having the amino acid sequence: Met Arg Ile Ala Val Thr Gly Ser Ile Ala Thr Asp His Leu Met Ala   1               5                  10                  15 Phe Pro Gly Arg Phe Gly Asp Gln Leu Ile Pro Asp Gln Leu Ala Arg              20                  25                  30 Val Ser Leu Ser Phe Leu Val Asp Gly Leu Glu Val Arg Arg Gly Gly          35                  40                  45 Val Ala Val Gly Ile Ala Phe Gly Leu Gly Arg Pro Gly Pro Thr Pro      50                  55                  60 Leu Leu Val Gly Ala Val Gly Asn Asp Phe Ala Asp Tyr Gly Thr Trp  65                  70                  75                  80 Pro Lys Glu His Gly Val Asp Thr Gly Gly Val Leu Val Pro Thr Glu                  85                  90                  95 His Gln Thr Ala Arg Phe Leu Cys Ile Thr Asp Arg Asp Ala Asn Gln             100                 105                 110 Ile Ala Ala Ser Tyr Thr Gly Ala Met Arg Glu Ala Arg Asp Ile Gly         115                 120                 125 Leu Arg Arg Thr Gly Ala Leu Pro Ala Pro Arg His Gly Leu Val Leu     130                 135                 140 Ile Cys Pro Asp Asp Pro Ala Ala Met Val Arg His Thr Ala Gln Cys 145                 150                 155                 160 Arg Glu Pro Gly Leu Pro Phe Val Ala Asp Pro Ser Gln Gln Leu Ala                 165                 170                 175 Arg Leu Glu Thr Asp Glu Val Arg Ala Leu Val His Gly Ala His Trp             180                 185                 190 Val Phe Thr Asn Glu Tyr Glu Ala Ala Leu Leu Leu Glu His Ser Gly         195                 200                 205 Trp Lys His Ser Glu Thr Leu Glu Arg Val Gly Ala Trp Val Thr Thr     210                 215                 220 Leu Gly Gly Ala Gly Val Arg Ile Glu Arg Ala Gly Glu Pro Pro Leu 225                 230                 235                 240 Thr Val Pro Ala Val Pro Asp Val Pro Val Val Asp Pro Thr Gly Ile                 245                 250                 255 Gly Ala Ala Phe Arg Ala Gly Phe Leu Ala Gly Ala Gly Arg Gly Leu             260                 265                 270 Ser Ile Val Ser Ala Ala Arg Leu Gly Cys Val Leu Ala Ala Arg Ala         275                 280                 285 Leu Gly Thr Val Gly Pro Ala Asp Leu Pro Asp Arg Ser Gly Gly Ser     290                 295                 300 Ala Arg His Gly Glu Gly Arg Val Arg Arg Gly Arg Gly Gly Ala Ala 305                 310                 315                 320 Arg Pro Arg Ala Gly Arg Pro His Met Thr Arg Pro Cys Pro Gly Ser                 325                 330                 335 Arg Arg Glu Pro Pro Ala Gly Arg Pro Ala Arg Ala Ala Ala Val Ile             340                 345                 350 Arg Arg Pro Gly Ala Gly Gly Pro Thr Ala Gly Gly Cys Arg  *         355                 360                 365

which is SEQ ID NO
 3. 3. A substantially pure MTHR from Streptomyces fradiae having the amino acid sequence: Met Arg Thr Thr Leu Arg Glu Ile Leu Gly Ser Gly Arg Leu Ser Phe   1               5                  10                  15 Ser His Glu Phe Phe Pro Pro Arg Thr Glu Ala Gly Thr Arg Thr Leu              20                  25                  30 Trp Asn Ala Ile Arg Arg Ile Glu Pro Leu Ala Pro Thr Phe Val Ser          35                  40                  45 Val Thr Tyr Gly Ala Gly Gly Ser Ser Arg Asp Arg Thr Val Glu Val      50                  55                  60 Thr Lys Arg Ile Ala Thr Asp Thr Thr Leu Arg Pro Val Ala His Leu  65                  70                  75                  80 Thr Ala Val Gly His Ser Val Ala Glu Leu Arg Arg Ile Ile Gly Gln                  85                  90                  95 Tyr Ala Asp Ala Gly Val Arg Asp Val Leu Ala Leu Arg Gly Asp Pro             100                 105                 110 Pro Gly Asp Pro Asn Ala Pro Trp Val Pro His Pro Glu Gly Leu Thr         115                 120                 125 His Ala His Glu Leu Val Ser Leu Val Arg Gly Ser Gly Gly Phe Gly     130                 135                 140 Val Gly Val Ala Ala Phe Pro Glu Arg His Pro Arg Ser Pro Asp Trp 145                 150                 155                 160 Asp Ser Glu Ile Arg His Phe Val Arg Lys Cys Arg Ala Gly Ala Asp                 165                 170                 175 Tyr Ala Ile Thr Gln Met Phe Phe Arg Val Glu Asp Tyr Leu Arg Leu             180                 185                 190 Arg Asp Arg Val Ala Ala Ala Gly Cys Cys Thr Pro Val Ile Pro Gly         195                 200                 205 Ile Met Pro Ala Thr Asp Val Arg Gln Ile Ala Arg Phe Ala Glu Leu     210                 215                 220 Ser His Ala Thr Phe Pro Glu Gly Leu Ala Arg Arg Leu Glu Ala Ala 225                 230                 235                 240 Arg Gly Asn Pro Ala Glu Gly His Arg Ile Gly Val Glu Tyr Ala Thr                 245                 250                 255 Ala Met Ala Gly Arg Leu Leu Ala Glu Gly Ala Pro Gly Leu His Tyr             260                 265                 270 Ile Thr Leu Asn Arg Ser Thr Ala Thr Leu Glu Ile His Arg Asn Ile         275                 280                 285 Leu Gly Thr Pro Ala Pro Gly Ser Ala Arg Gln Val Leu Ala Ala Pro     290                 295                 300 Leu  * 305

which is SEQ ID NO
 5. 4. An isolated nucleic acid compound encoding the protein of claim
 1. 5. An isolated nucleic acid compound comprising a sequence encoding the protein of claim 1 or fragment thereof wherein said compound has a sequence selected from the group consisting of: (a) residues 986 through 2209 of SEQ ID NO:1; (b) residues 986 through 2209 of SEQ ID NO:6 (c) a nucleic acid compound complementary to (a) or (b); and (d) a fragment of (a), (b), or (c) that is at least 18 base pairs in length and which will selectively hybridize to genomic DNA encoding SEQ ID NO.
 2. 6. An isolated nucleic acid compound of claim 5 wherein the sequence is (a) or a sequence complementary to (a).
 7. An isolated nucleic acid compound of claim 5 wherein the sequence of said compound is (b) or a sequence complementary to (b).
 8. An isolated nucleic acid compound encoding the protein of claim
 2. 9. An isolated nucleic acid compound comprising a sequence encoding the protein of claim 2 or fragment thereof wherein said compound has a sequence selected from the group consisting of: (a) residues 2241 through 3341 of SEQ ID NO:1; (b) residues 2241 through 3341 of SEQ ID NO:6 (c) a nucleic acid compound complementary to (a) or (b); and (d) a fragment of (a), (b), or (c) that is at least 18 base pairs in length and which will selectively hybridize to genomic DNA encoding SEQ ID NO.
 3. 10. An isolated nucleic acid compound of claim 9 wherein the sequence is (a) or a sequence complementary to (a).
 11. An isolated nucleic acid compound of claim 9 wherein the sequence of said compound is (b) or a sequence complementary to (b).
 12. An isolated nucleic acid compound encoding the protein of claim
 3. 13. An isolated nucleic acid compound comprising a sequence encoding the protein of claim 3 or fragment thereof wherein said compound has a sequence selected from the group consisting of: (a) residues 3338 through 4255 of SEQ ID NO:4; (b) residues 3338 through 4255 of SEQ ID NO:6 (c) a nucleic acid compound complementary to (a) or (b); and (d) a fragment of (a), (b), or (c) that is at least 18 base pairs in length and which will selectively hybridize to genomic DNA encoding SEQ ID NO.
 5. 14. An isolated nucleic acid compound of claim 13 wherein the sequence is (a) or a sequence complementary to (a).
 15. An isolated nucleic acid compound of claim 13 wherein the sequence of said compound is (b) or a sequence complementary to (b).
 16. An isolated nucleic acid compound encoding the SAM operon from Streptomyces fradiae , wherein said compound is SEQ ID NO.1 or SEQ ID NO.6.
 17. A vector comprising an isolated nucleic acid compound of claim 16 wherein said compound is SEQ ID NO.1.
 18. A vector comprising an isolated nucleic acid compound of claim 5 in operable linkage to a promoter sequence.
 19. A vector comprising an isolated nucleic acid compound of claim 9 in operable linkage to a promoter sequence.
 20. A vector comprising an isolated nucleic acid compound of claim 13 in operable linkage to a promoter sequence.
 21. A host cell containing a vector of claim
 17. 22. A host cell containing a vector of claim
 18. 23. A host cell containing a vector of claim
 19. 24. A host cell containing a vector of claim
 20. 25. A method for constructing a recombinant host cell having the potential to express SEQ ID NO:2, SEQ ID NO.3, and SEQ ID NO.5 said method comprising introducing into a host cell by any suitable means a vector of claim
 16. 26. A method as in claim 25 wherein said host cell is an Actinomycete.
 27. A method for expressing SEQ ID NO:2, SEQ ID NO.3 and SEQ ID NO.5 in the recombinant host cell of claim 26, said method comprising culturing said recombinant host cell under conditions suitable for gene expression.
 28. A method for synthesizing S-adenosylmethionine, comprising the steps of: a) admixing a suitable amount of the following reagents: i) a substantially pure SAM synthetase as claimed in claim 1, ii) ATP; iii) methionine; and b) purifying said S-adenosylmethionine by any suitable means.
 29. A method for producing S-adenosylmethionine comprising the steps of: a) preparing a crude extract from the recombinant cells of claim 22; b) adding to said extract suitable amount of ATP and methionine; and c) recovering said S-adenosylmethionine by any suitable means.
 30. A method for producing S-adenosylmethionine comprising the steps of: a) culturing the host cell of claim 21 under conditions suitable for synthesis of S-adenosylmethionine, and b) recovering S-adenosylmethionine from said host cell by any suitable means. 